As we all know, bio-terrorism in America is a reality. However in addition to the Category A agents like anthrax, Yersinia pestis and smallpox, which are difficult to safely grow and disseminate, exist the Category B agents that could be used to infect our food or water supply. These organisms include bacterial pathogens, protozoa, and viruses. In addition to these natural pathogenic organisms they could also be genetically engineered to increase their virulence or resist standard antibiotic treatments. Therefore new methods for rapid food and waterborne pathogen detection is greatly needed especially if it can also be used to identify drug sensitivity within these organisms. This program will develop such tests. In addition to bio-terrorism concerns, food-borne diseases cause 76-million illnesses, including 325,000 hospitalizations and 5,000 deaths in the US each year. More than 200 known diseases are transmitted through food and drink. Traditional detection of food-borne pathogens has relied on microbiologic techniques. These standard culture methods recommended by the AOAC require between 5 to 7 days to complete. Meanwhile, the product is held for shipment, increasing storage costs and reducing the product shelf life. In the case of our water supply, Public Health Service warnings are released when problems are identified, usually after the product is already released for public consumption. Consequently, rapid methods for food and water pathogen detection are greatly needed to protect the public health. The overall goal of this program is to develop an integrated isothermal DNA amplification and detection system capable of rapidly identifying a variety of food and waterborne pathogens. All of the NIAID Biodefense Category B food and waterborne bacterial pathogens will be detected in this program. A single integrated system capable of isothermal amplifying and detecting all of these organisms in a closed device is proposed.